Branched-DNA (bDNA) signal-amplification technology has been used extensively in a microwell format to detect and quantify specific nucleic acid sequences. Urdea et al. (2000) Branched-DNA (bDNA) Technology. In Kessler C., ed., Nonradioactive Analysis of Biomolecules, New York, Springer-Verlag:388–395. Inherently quantitative and highly reproducible, bDNA can be applied to the detection of any nucleic acid target for which a sequence is known without the use of radioactive probes.
A number of bDNA assays have been developed for the quantification of viral nucleic acids. These include: human immunodeficiency virus type 1 (HIV-1) RNA, Kern et al. (1996) J Clin Microbiol 34:3196–3203; simian immunodeficiency virus (SIV) RNA, Sodora et al. (1998) AIDS Res Hum Retroviruses 14:171–181; hepatitis B virus (HBV) DNA, Hendricks et al. (1995) Am J Clin Pathol 104:537–546; hepatitis C virus (HCV) RNA, Detmer et al. (1996) J Clin Microbio 34:901–907; hepatitis G virus (HGV) RNA, Brandhagen et al. (1999) Am J Gastroenterol 94:1000–1005; and cytomegalovirus (CMV) DNA, Chernoff et al. (1997) J Clin Microbiol 35:2740–2744.
More recently, bDNA technology has been used to detect and measure expression of cellular mRNAs, including: cytokines, Breen et al. (1997) Cell Immunol 178:91–98 and Shen et al. (1998) J Immunol Methods 215:123–134, progesterone and estrogen receptors, Nargessi et al. (1998) Breast Cancer Res Treat 50:47–55 and Nargessi et al. (1998) Breast Cancer Res Treat 50:57–62; insulin, Wang et al. (1997) Proc Natl Acad Sci USA 94:4360–4365; glucokinase, Cabrera-Valladares et al. (1999) Endocrinology 140:3091–3096; c-fos, Shyamala et al. (1999) Anal Biochem 266:140–147; and aP2, Burris et al. (1999) Mol Endocrinol 13:410–417. All of these bDNA assays were developed to measure nucleic acids in serum, plasma or cell lysates. None of these studies, however, suggests or discloses a bDNA assay for the detection of nucleic acids in morphologically intact cells or tissues.
In contrast, an in situ hybridization (ISH) assay would necessarily have the capability to detect specific nucleic acid sequences in morphologically intact cells or tissues. ISH methods have been improved since the general concept was introduced by Pardue and Gall over twenty years ago. Pardue et al. (1969) Proc Natl Acad Sci USA 64:600–604. For example, use of non-isotopic probes has eliminated the inherent problems associated with radioactive ISH methods such as long turn-around times, risk of exposure to radioactivity and waste disposal.
In addition, the incorporation of various target and signal-amplification systems has improved ISH sensitivity. Notwithstanding these advances in ISH methods, however, a number of challenges must still be overcome. These challenges include developing sensitive and specific target detection, ensuring precise co-localization of signal and target, preserving target sequences, and preserving cellular and tissue morphology. In addition, ISH methods must address a number of practical concerns, including ease-of-use, reproducibility and amenability to quantification, amenability to automation, versatility and timely completion.
Catalyzed reporter deposition tyramide signal amplification (CARD/TSA) is one ISH method that has been proposed to address some of these challenges and concerns. Some studies have shown that the CARD/TSA ISH method can detect 1–2 copies of HPV-16 DNA in SiHa cells. Siadat-Pajouh et al. (1994) J Histochem Cytochem 42: 1503–1512 and Adler et al. (1997) Histochem Cell Biol 108:321–324.
One study has shown that it is possible to adapt bDNA technology to an ISH format for the detection of mRNA. Cao et al. (1998) Proceedings of the American Association for Cancer Research, 89th meeting, New Orleans, La. and Antao et al. (1999) In Situ Hybridization Using the bDNA Technology. In Patterson, B. K., ed., Techniques in Quantification and Localization of Gene Expression, Boston, Birkhauser Press:81–93. The assay described in this study, however, has relatively low sensitivity.
Thus, there remains a need in the art to provide highly sensitive methods for in situ detection of nucleic acid analytes in biological samples. The present invention satisfies these and other needs in the art.